[NOT APPLICABLE]
[NOT APPLICABLE]
This invention relates to novel ophthalmic compositions useful for conditioning and/or cleaning and/or disinfecting contact lenses. In particular, the present invention provides ophthalmic compositions that contain one or more indolicidin antimicrobial polypeptides and typically have a low halide ion concentration.
During normal use, contact lenses are xe2x80x9csoiledxe2x80x9d or xe2x80x9ccontaminatedxe2x80x9d with a wide variety of compounds that immediately or ultimately degrade lens performance. For example, while being worn on the eye, a contact lens is liable to be contaminated with biological materials such as protein or lipid that are found in the tear fluid and that adheres to the surfaces of the contact lens. In handling or cleaning the contact lens, sebum (skin oil) or cosmetics or other materials adhering to the hands of the user tend to soil the contact lens. In addition, particularly as contact lenses accumulate organic contaminants they promote the growth on their surface of a wide range of microbes.
If the contact lens is worn on the eye with such contaminants on its surface, the contact lens suffers from deteriorated water wettability or hydrophilicity and lowered oxygen permeability, causing considerable discomfort to the lens wearer. In addition, the lens wearer may suffer from deterioration in his eyesight, pain in the eye, hyperemia or congestion of the eye, due to the continuous wearing of the contaminated contact lens. In addition, a contaminated lens can become immunogenic stimulating an adverse immune response on the eye and associated tissues. In view of this, it is important to remove the soil debris adhering to the contact lens surfaces and to disinfect the lens for safe and comfortable wearing of the contact lens on the user""s eye.
In view of these, and other, factors effecting lens performance, a care regimen for contact lenses typically involves various functions, such as regularly cleaning the lens with a contact lens solution. Rinsing of the contact lens is generally required following cleaning to remove loosened debris and to remove potential irritants in the cleaning solution. Additionally, the regimen may include treatment to disinfect the lens, treatment to render the lens surface more wettable prior to insertion in the eye, or treatment to condition (e.g., lubricate or cushion) the lens surface so that the lens is more comfortable in the eye. As a further example, a contact lens wearer may need to rewet the lens during wear by administering directly in the eye a solution commonly referred to as rewetting drops.
Separate solutions may be provided for the individual segments of the care regimen. For convenience purposes, multipurpose contact lens solutions have gained popularity, i.e., solutions which can be used for several segments of the care regimen.
As an example, multipurpose contact lens solutions that can be used for cleaning, storage and conditioning of contact lenses have been suggested (see, e.g., U.S. Pat. No. 5,141,665 (Sherman) discloses a cleaning, conditioning, storing and wetting system for rigid gas permeable contact lenses. The system is described as including: (1) a cleaning, conditioning and storing solution; and (2) a separate wetting solution, where both solutions include a disinfectant or preservative. Lenses treated with the first solution are rinsed and then wet with the separate wetting solution prior to insertion in the eye.
Multipurpose contact lens solutions that effectively clean a contact lens, and can also be used to treat the lens immediately prior to insertion of the lens in the eye, are much more challenging multipurpose solutions to develop. Conventional surface active agents having good cleaning activity for contact lens deposits, as well as various other components such as antimicrobial agents included as a preservative or disinfectant, tend to be irritating to the eye. Accordingly, new ophthalmic compositions, particularly multipurpose formulations, are desirable.
In recent years, researchers have come to recognize that many organisms use peptides as part of their host defense systems. These organism include a full range of species from prokaryotes to humans. The antimicrobial peptides can be subdivided into a number of groups based on their amino acid content, structure and source. Several reviews of several classes of these peptides have been recently published (See, for example, Lehrer and Ganz (1966) Annal. N.Y. Acad. Sci., 797:228-239; Maloy and Kari (1995) Biopolymers, 37:105-122).
Antimicrobial peptides have common structural features, including a net cationic charge due to the presence of multiple charged residues (Arg, Lys), the presence of multiple cysteine residues, and in most cases the ability to form amphipathic structures. These properties are important for the mechanism of action that is currently thought to involve a non-receptor-mediated interaction with the anionic phospholipid bilayer of the target cell, followed by incorporation of the peptide into the membrane, and a resulting disruption of the membrane structure.
The spectrum of activity of host defense peptides is very broad, killing many species of bacteria, protozoa, fungi, and virally infected cells and even cancer cells (Maloy and Kari (1995) Biopolymers, 37:105-122). Moreover, these peptides have a low toxicity on most healthy mammalian cells and tissues. It is thought that the peptides are preferentially selective for prokaryotic membranes because of the lipid composition of the membrane. In particular, there is a higher concentration of anionic phospholipids in the outer leaflet of bacteria than in normal eukaryotic membranes, and cholesterol is present in mammalian membranes but not in bacterial membranes. Whatever the reason for the preference, the selectivity of antimicrobial peptide for prokaryotes compared to eukaryotes, these peptides are ideal components in products intended for human use.
While attempts have been made to utilize antimicrobial polypeptides in ophthalmic solutions, to date such efforts have been largely unsuccessful. For example, Sousa et al. (1996) CLAO J., 22(2):114-117, showed that the use of a synthetic cecropin analog in combination with various ophthalmic solutions failed to be highly effective in disinfecting, particularly when used by itself. Cullor et al. (1990) Arch. Ophthalmol., 108: 861-864 examined the in vitro efficacy of rabbit defensins, NP-1 and NP-5, against ocular pathogens isolated from cases of severe ulcerative keratitis in humans and horses. Unfortunately, these antimicrobial peptide-containing compositions were found to have toxicity to human eye tissue. Similarly U.S. Pat. No. 5,549,894 describes ophthalmic compositions containing D-enantiomeric peptides, such as cecropins, magainins and defensins. These polypeptides, however are toxic and eye care products containing these peptides can not be made in commercial quantities, as the peptides must be chemically synthesized and can not be made using biological expression systems.
This invention provides novel ophthalmic solutions based on the use of indolicidins as antimicrobial agents. In particular, the solutions of this invention are self-preserving and require no additional preservatives or disinfectants. The solutions are well suited for ophthalmic use and are effective in cleaning, disinfecting, and sterilizing the contact lens upon exposure to the composition without the need for physical or thermal treatment of the lens. Moreover, because indolicidins are safe for topical application to the eye, the solutions enable immediate application of the contact lens to the eye without the need for neutralization, deactivation, or washing any of the compositions"" components. In addition, the compositions have extremely long shelf life at room temperature.
In particular, it was a discovery of this invention that indolicidins are inactivated by the presence of chloride in typical buffer systems (e.g., phosphate buffered saline (PBS)), but can act as highly effective antimicrobial compounds when formulated either in solutions containing a low concentration of halide ion, or when formulated in a Good""s buffer (e.g., Tris(hydroxymethyl)aminomethane (TRIS).
Embodiments of the invention include an ophthalmic composition for storing, cleaning, or disinfecting a contact lens, the composition comprising an indolicidin in an antimicrobially effective amount and a buffer compatible with application to a mammalian eye, wherein the buffer has a halide ion concentration of less than 0.85%. The composition may additionally comprise boric acid or a borate salt present in a microbistatic amount, a poloxamer, a chelating agent, or a divalent cation.
In a preferred embodiment, the composition comprises indolicidin, wherein the indolicidin is present in a concentration ranging from 2 xcexcg/ml to 100 xcexcg/ml; and a buffer comprising a sodium phosphate or potassium phosphate in a concentration ranging from about 1 mM to about 10 mM; a boric acid or borate salt in a concentration ranging from about 0.1% to about 5%; a poly(oxyethylene)-poly(oxypropylene) block copolymer in a concentration ranging from about 0.25 to about 0.5%; a chelator in a concentration ranging from about 0.01% and about 0.5%; and a divalent cation in a concentration ranging from about 5 mM to about 50 mM.
An alternative composition of the invention for storing, cleaning, or disinfecting a contact lens comprises an indolicidin and a Good""s buffer. The composition may additionally comprise boric acid or a borate salt present in a microbistatic amount, a surfactant (preferably a poloxamer), a chelating agent, or a divalent cation.
In additional embodiments, the invention comprises a multipurpose solution for the care of a contact lens, the solution comprising indolicidin and a buffer wherein the solution is suitable for two or more actions, including contact lens disinfection, contact lens storage, contact lens cleaning, contact lens conditioning and rehydrating, contact lens moistening, and contact lens lubricating. The solution comprises indolicidin and a buffer compatible with application to a mammalian eye wherein the halide ion concentration of the buffer is less than 0.85%. Alternatively, the solution may comprise indolicidin and a Good""s buffer. Such a multipurpose solution may additionally comprise a demulcent and a poloxamer.
The invention includes a method of disinfecting a contact lens, the method comprising contacting the contact lens with a composition of the invention. Further, a contact lens storage system is included in the invention, which comprises a container containing an opthalmic composition of the invention.
Alternatively, the invention may comprise a method of packaging a contact lens, the method comprising sealing a contact lens in a container with an ophthalmic composition of the invention or a method of disinfecting a contact lens storage vessel comprising contacting the storage vessel with a composition of the invention.
Definitions
The term Good""s buffer refers to a buffer of the type described by Good et al. (1966) Biochem., 5: 467-477. Preferred Good""s buffers include tris(hydroxymethyl)aminomethane (TRIS), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 2,4-(2-hydroxyethyl)-1-piperazinyl ethanesulfonic acid (HEPES), 3,4-(2-hydroxyethyl)-1-piperazinyl propanesulfonic acid (EPPS), N-tris(hydroxymethyl) methylglycine (Tricine), N,N-bis (2-hydroxyethyl)glycine (Bicine), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), with TRIS being most preferred.
The term xe2x80x9cantimicrobialxe2x80x9d is used herein to refer to the ability of a compound, i.e. an indolicidin, to decrease the population of microscopic flora and/or fauna on a surface, e.g., on a contact lens surface. Antimicrobial activity includes bacteriostatic or antibacterial activity, antifungal activity, antialgal activity, and the like. An antimicrobial need not eliminate all microbes, but simply decreases the viable population on the treated surface.
Similarly, xe2x80x9cantimicrobial activityxe2x80x9d refers to the ability of a compound to inhibit or irreversibly prevent the growth of a microorganism. Such inhibition or prevention can be through a microbicidal action or microbistatic inhibition.
xe2x80x9cMicrobicidal inhibitionxe2x80x9d as used herein refers to the ability of the antimicrobial to kill or irrevocably damage the target organism.
xe2x80x9cMicrobistatic inhibitionxe2x80x9d as used herein refers to the ability of the microbistatic or antimicrobial compound to inhibit or to retard the growth of the target organism without causing death. Microbicidal or microbistatic inhibition can be applied to either an environment either presently exhibiting microbial growth (i.e., therapeutic treatment) or an environment at risk of supporting such growth (i.e., prevention or prophylaxis).
xe2x80x9cBroad spectrum antimicrobial activityxe2x80x9d refers to the ability of a compound to inhibit or prevent the survival or growth of various prokaryotic and eukaryotic microorganisms including, for example, protozoans such as Giardia lamblia, fungi such as Cryptococcus, various genera of bacteria such as Escherichia, Salmonella and Staphylococcus, and enveloped viruses. Antimicrobial activity can occur through a microbicidal or a microbistatic inhibition.
xe2x80x9cMicrobicidal inhibitionxe2x80x9d refers to the ability of a compound to reduce or inhibit the survival of a microorganism by killing or irreversibly damaging it, whereas the term xe2x80x9cmicrobistatic inhibitionxe2x80x9d refers to the ability of a compound to inhibit the growth of a target microorganism without killing it. A compound having microbicidal or microbistatic inhibition can be applied to an environment that presently allows for the survival or growth of a microorganism (i.e., therapeutic treatment) or to an environment at risk of supporting such survival or growth (i.e., prevention or prophylaxis).
xe2x80x9cAntimicrobial selectivityxe2x80x9d refers to the relative amount of antimicrobial activity of an analog as compared to its cytolytic activity against normal cells in a subject.
The term xe2x80x9cophthalmic solutionxe2x80x9d refers to a solution that forms a stock solution for the preparation of solutions for the packaging, shipping, storage, cleaning, maintenance, use, or rehydration of contact lenses or is useful itself for one or more of these purposes.
As used herein, the terms xe2x80x9cophthalmically acceptable compositionxe2x80x9d or xe2x80x9ccompatible with application to an eyexe2x80x9d means a composition which can be placed into a human eye without causing any substantial discomfort, damage, or harm.
The terms xe2x80x9cstablexe2x80x9d or xe2x80x9cstabilityxe2x80x9d when used with respect to an indolicidin refer to the retention of antimicrobial activity (e.g., as measured by a minimal inhibitory concentration (MIC) assay, see, e.g., Example 10). When the indolicidin or indolicidin-based formulation is unstable, it looses activity with time. The indolicidin or indolicidin-based composition is deemed unstable over a selected period time under particular storage conditions (e.g., at room temperature in a particular type of receptacle), when the indolicidin or indolicidin-based composition shows a significant diminution in microbicidal activity against one or more selected test organisms (e.g., Pseudomonas, sp.). A significant diminution is typically at least a two percent diminution, preferably at least a 5 percent diminution, more preferably at least a 10% diminution and most preferably at least a 20% diminution in activity. Conversely, the indolicidin or indolicidin-based composition is said to be stable over the particular time period/storage conditions, when there is less than a 20%, preferably less than a 10%, more preferably less than a 5%, and most preferably less than a 2% or 1% decrease in activity as determined by a MIC assay.
The term xe2x80x9cweight percentxe2x80x9d or xe2x80x9cwt %xe2x80x9d as used herein refers to the number of grams of material per 100 ml of solution.
The term xe2x80x9clens disinfectionxe2x80x9d refers to a reduction or elimination of microbial organisms present on a contact lens.
The term xe2x80x9clens storagexe2x80x9d, refers either to the short term storage e.g., on the order of hours, days or weeks, by a user, e.g, when not wearing the lens, or long term storage, e.g., on the order of days, weeks, months, or years, as in the storage prior to sale or first use of the lens.
xe2x80x9cLens cleaningxe2x80x9d refers to a reduction and/or elimination of contaminants on the surface of a contact lens. Such contaminants include, but are not limited to lipids, fats, oils, proteins, or microbial organisms.
xe2x80x9cLens conditioning and/or rehydratingxe2x80x9d refers to the addition of water to a lens, e.g., a soft contact lens thereby increasing the water content of the lens.
xe2x80x9cLens lubricatingxe2x80x9d refers to the addition of water, surfactants, or demulcents to decrease the coefficient of friction between a lens and the surface of an eye. Such lubricating can be by treatment of the lens prior to or after insertion into the eye.
The term xe2x80x9cmammalian eyexe2x80x9d is used to refer the to eye of any animal in the order mammalia. Such animals include, but are not limited to horses, cats, dogs, rabbits, mice, goats, sheep, non-human primates and humans. Thus, the solutions are contemplated for use in veterinary applications as well as human use. In preferred embodiments, the mammalian eye is a xe2x80x9chealthyxe2x80x9d eye. However, under certain circumstances, the eye may be unhealthy, e.g., infected, inflamed, or otherwise irritated.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d, or xe2x80x9cproteinxe2x80x9d are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
xe2x80x9cAmino acidxe2x80x9d is used in its broadest sense to include naturally occurring amino acids as well as non-naturally occurring amino acids including amino acid analogs. In view of this broad definition, one skilled in the art would know that reference herein to an amino acid includes, for example, naturally occurring proteogenic (L)-amino acids, (D)-amino acids, chemically modified amino acids such as amino acid analogs, naturally occurring non-proteogenic amino acids such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. As used herein, the term xe2x80x9cproteogenicxe2x80x9d indicates that the amino acid can be incorporated into a protein in a cell through a metabolic pathway.
Indolicidin (SEQ ID NO: 1) is a thirteen amino acid polypeptide that has a high tryptophan content, exhibits broad spectrum antimicrobial activity and has antimicrobial selectivity. As used herein, indolicidins should be understood to include both native indolicidin and analogs thereof. Indolicidins and their use in antimicrobial formulations are described in U.S. Pat. No. 5,547,939. Indolicidins useful in accordance with the present invention have similar tryptophan content, antimicrobial activity and antimicrobial selectivity to native indolicidin (see Table 2). In general, indolicidins useful in accordance with the present invention have the general structure H2N-I-L-P-W-K-W-P-W-W-P-W-X (SEQ ID NOS:14 and 15), where X designates one or two independently selected amino acids. Indolicidins typically contain twelve or thirteen amino acids (although the indolicidins of this invention can range in length up to about 45 amino acids) and are tryptophan-rich. Indolicidin, for example, has a tryptophan content of about 38 percent (5/13 residues). Indolicidins are further characterized by having substantially the same sequence as naturally occurring indolicidin. As used herein, the term xe2x80x9csubstantially the same sequencexe2x80x9d means that the peptide sequence of an indolicidin analog has at least 60%, preferably 70%, more preferably 80%, and most preferably 90%, 95%, or 98% sequence identity with the sequence of indolicidin (SEQ ID NO: 1). Thus, a limited number of modifications can be made to the indolicidin peptide sequence to obtain indolicidins that have a desirable antimicrobial selectivity such as increased antimicrobial activity or decreased hemolytic activity as compared to naturally occurring indolicidin. For example, an indolicidin analog can have the same peptide sequence as indolicidin but can be modified, for example, by containing a C-terminal reactive group other than an amide, which is found in naturally occurring indolicidin (see, for example, (SEQ ID NO: 5).
xe2x80x9cTryptophan-rich peptidexe2x80x9d means a peptide having at least about 25% of its residues consisting of tryptophan.
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5: 151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA, 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
The term xe2x80x9cconservative substitutionxe2x80x9d is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (e.g., antimicrobial activity) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K)
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)